A. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly it relates to latent TGFxcex2 binding protein (LTBP) genes, compositions and methods of use.
B. Description of the Related Art
1. TGF-xcex2
Five TGF-xcex2 family members, which share 66-82% sequence identity, have been identified (Kingsley, 1994). Whereas TGF-xcex21 was cloned from a cDNA library derived from human placenta, TGF-xcex22 was subsequently purified from several mammalian cells and tissues, and TGF-xcex23, -xcex24, and -xcex25 were cloned by low stringency hybridization from mammalian, avian and amphibian cDNA libraries, respectively. Peptide growth factors/cytokines have also been identified that share sequence homology (xe2x89xa640%) with the TGF-xcex2s (collectively, the TGF-xcex2s plus these other cytokines make up the TGF-xcex2 superfamily). A unifying feature of the biology of these other cytokines (i.e., the Mullerian inhibiting substance, bone morphogenetic proteins, growth and differentiation factors, activin/inhibin, Drosophila decapentaplegic complex, and amphibian Vg1 protein) is the ability to regulate developmental processes. In every case where information is available, superfamily members are synthesized as larger precursors that are processed at endoproteolytic cleavage motifs, and they terminate with the sequence C-X-C-X. The three dimensional crystal structure of the TGF-xcex22 homodimer was recently reported (McDonald and Hendrickson, 1993). This work has led to the interesting and novel suggestion that TGF-xcex2 is related to certain peptide growth factors (e.g., NGF, PDGF, v-SIS) in a way that could not have been predicted from the deduced amino acid analysis.
2. Latent TGF-xcex2 Complexes
Many cell types produce TGF-xcex2, and almost all cells bind TGF-xcex2 with affinities in the picomolar rangexe2x80x94e.g., the type I and type II TGF-xcex2 cell surface receptors (glycoproteins of 53 and 75 kDa, respectively) are present in essentially all cells (Miyazono et al., 1994). Thus, TGF-xcex2 has powerful effects on most cell types, and cytokines such as TGF-xcex2 are thought to exert broad control the tissue remodeling that occurs during development, wound repair, and other situations (Sporn et al., 1986; Moses et al., 1990). (For a comprehensive all review of TGF-xcex2 effects, see Roberts and Sporn, 1990). For example, TGF-xcex2 was initially identified as a factor that stimulated the anchorage independent growth of rodent fibroblasts (Assoian et al., 1983; Frolik et al., 1983; Roberts et al., 1983). It is now known, however, that TGF-xcex2 acts as a potential growth inhibitor for most cells, i.e., epithelial, endothelial, and hematopoietic progenitor cells; both stimulates and inhibits cellular differentiation; induces extracellular matrix production by stimulating the expression of matrix macromolecules, stimulating the expression of matrix protease inhibitors, and decreasing the expression of matrix degrading proteases; inhibits the functional activities of immune cells; induces the chemotaxis of fibroblasts, macrophages, and smooth muscle cells; induces angiogenesis in vivo; inhibits endothelial migration; induces the expression of cell surface receptors for other cytokines (e.g., the EGF receptor); promotes the healing of incisional wounds; inhibits osteoblast proliferation in vitro; and induces new bone formation in vivo.
A molecular explanation for these complex (and, at times, conflicting) effects is not yet available, but hypotheses do exist. Sporn et al. (1986) have suggested, for example, that the ability of TGF-xcex2 to stimulate or inhibit the proliferation of mesenchymal cells depends on the state of cellular differentiation and the entire set of growth factors operant in that cell population. As such, the xe2x80x9cbiological meaningxe2x80x9d of TGF-xcex2 signal transduction depends on the context (i.e., availability and presentation) of other growth factors present in the local environment: Fischer rat 3T3 cells transfected with a myc gene and incubated with TGF-xcex2 and PDGF proliferate in soft agar, whereas the same cells in the presence of TGF-xcex2 and EGF fail to grow (Roberts et al., 1985).
Whatever the mechanism, the autocrine and paracrine activities of TGF-xcex2 clearly must be regulated with precision. One regulatory strategy involves the temporal and spatial control of TGF-xcex2 gene expression. A second strategy involves the production and storage of TGF-xcex2 as a latent complex that is activated only under certain physiological and pathological conditionsxe2x80x94e.g., tissue morphogenesis and remodeling, and wound healing. TGF-xcex21 can be isolated from serum and from most tissues as a latent complex (Pircher et al., 1986; Miyazono et al., 1988; Wakefield et al., 1988). In this regard, the latent complex has been purified from human platelets and characterized in detail (Miyazono et al., 1988). Following a 6-step protocol, the purified complex yielded protein bands of Mr 25,000 and 210,000 on SDS-PAGE under nonreducing conditions. After reduction, the 25 kDa band was shown to consist of subunits of Mr 12,500. On the other hand, the 210 kDa band consisted of a Mr 40,000 subunit and Mr 125-160,000 subunit.
TGF-xcex2 is also secreted from several producer cell lines in culture as a latent complex of 235 kDa (Gentry et al., 1987). TGF-xcex21 is initially synthesized in vitro as a 390 amino acid precursor that consists of a signal peptide, an amino-terminal propeptide, and the mature growth factor. Two precursor chains associate to form a disulfide-bonded dimer with latent activity. Homodimers occur most commonly, but heterodimers may also form (Ogawa et al., 1992). The full length dimer is cleaved at a endoproteolytic cleavage motif, but the propeptide dimer (i.e., the latency associated peptide or LAP) and the mature growth factor dimer typically remain non-covalendy associated. The mature TGF-xcex2 dimer is now known to be the 25 kDa band identified after nonreducing SDS-PAGE of the purified latent complex from platelets. In addition, LAP is known to be a component of the 210 kDa band identified after nonreducing SDS-PAGE of the purified latent complex from platelets (i.e., LAP has been shown to consist of two of the 40 kDa subunits).
Together LAP and the mature TGF-xcex2 dimer form the small latent complex. As demonstrated in platelets, small latent complexes may be associated with additional high molecular weight proteins, the best characterized of which is the latent TGF-xcex2 binding protein or LTBP (Kanzaki et al., 1990). (LTBP has been shown to be the 125-160 kDa subunit of the purified latent complex from platelets). Latent TGF-xcex2 complexes that contain LTBP are also known as large latent complexes. In contrast to platelet LTBP, the LTBP produced by fibroblasts typically is a 190 kDa polypeptide. The smaller size of platelet LTBP may be due to proteolytic processing or alternative splicing (Kanzaki et al., 1990; Tsuji et al., 1990).
3. LAP and Latency TGF-xcex2 latency results in part from the non-covalent association of the propeptide dimer and the mature TGF-xcex2 dimer (Pircher et al., 1984; Gentry et al., 1988; Wakefield et al., 1989). A cDNA for the TGF-xcex21 precursor was expressed in Chinese Hamster Ovary (CHO) cells, which do not express LTBP (Gentry et al., 1988), and almost all TGF-xcex2 activity recovered from the medium of transfected cells was latent. Use of deletion constructs has demonstrated that synthesis of biologically active TGF-xcex21 can proceed only from the first ATG codon, implicating LAP in the proper assembly of the small latent complex in these cells. Taken together, these studies indicate that LAP is sufficient to achieve the latent state. More recent studies have shown that carbohydrate structures within LAP make an important contribution to the latent state. For example, treatment of the latent form of TGF-xcex21 with endoglycosidase F led to activation of TGF-xcex2 (Miyazono and Heldin, 1989). (The structure of the mature TGF-xcex2 dimer was not affected by enzyme treatment). In particular, sialic acid residues seemed to be important, as treatment of the purified latent complex with sialidase was also able to activate TGF-xcex2 from the latent state.
4. Modulation of Latency
Latent complexes must be dissociated to activate mature TGF-xcex2, art dissociation is considered to be a critical step in governing TGF-xcex2 effects (Twardzik et al., 1990; Sato et al., 1993). Dissociation by chemical treatment of the latent complex purified from platelets has been investigated (Miyazono et al., 1990). Incubation of the purified complex under conditions of varying pH revealed that TGF-xcex2 activity was unmasked at values below pH 3.5 and above pH 12.5. Incubation of latent TGF-xcex2 in 0.02% SDS or 8 M urea also effectively unmasked TGF-xcex2 activity, but incubation in 5 M NaCl did not. Wakefield et al. (Wakefield et al., 1989) have reported that, after activation, TGF-xcex21 and LAP reassociate in a time- and concentration-dependent manner under neutral, nondenaturing conditions. These results are consistent with the idea that the mature TGF-xcex2 dimer is non-covalently associated with LAP.
Latent TGF-xcex2 complexes are also dissociated by the action of certain enzymes. For example, latent TGF-xcex2 is activated by plasmin, which disrupts the structure of the large latent complex (Lyons et al., 1988; Taipale et al., 1995). Similar data exist for other enzymes, e.g., cathepsin D, mast cell chymase, leukocyte elastase, and the glycosidases. Recently, osteoclast-derived cells were shown to be capable of activating latent TGF-xcex2 in vitro (Oreffo et al., 1989). Osteoclast activation is of particular interest because of the hypothesis that TGF-xcex2 serves as a link between bone turnover and formation during bone remodeling (Centrella et al., 1991). The mechanism of TGF-xcex2 activation by osteoclasts is not known at present, but it is reasonable to think that local alteration of pH due to action of proton pumps in the osteoclast plasma membrane or the release of osteoclast-derived proteases may be involved in the activation process. Related to these observations, activated macrophages (as might be found at a wound site or during tissue morphogenesis) secrete sialidase and other proteases (Pilatte et al., 1987), and they can lower the local pH to 4.0 (Silver et al., 1988), both of which could contribute to TGF-xcex2 activation in vivo. As mentioned above, acidification weakens the non-covalent interaction between LAP and the mature TGF-xcex2 dimer (Wakefield et al., 1989).
The present invention concerns in an overall and general sense novel DNA segments and recombinant vectors encoding LTBP-2 or LTBP-3, and the creation and use of recombinant host cells through the application of DNA technology, that express LTBP-2 or LTBP-3 gene products. As such, the invention concerns DNA segment comprising an isolated gene that encodes a protein or peptide that includes an amino acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:2 or SEQ ID NO:4. These DNA segments are represented by those that include a nucleic acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:1 or SEQ ID NO:3.
Compositions that include a purified protein that has an amino acid sequence essentially as set forth by the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 are also encompassed by the invention.
The TGF-xcex2s represent a family of structurally related molecules with diverse effects on mammalian cell shape, growth, and differentiation (Roberts and Sporn, 1990). Initially synthesized as a precursor consisting of an amino-terminal propeptide followed by mature TGF-xcex2, two chains of nascent pro-TGF-xcex2 associate in most tissues to form a Mr xcx9c106,000 inactive disulfide-bonded dimer. Homodimers are most common, but heterodimers have also been described (Cheifetz et al., 1987; Ogawa et al., 1992). During biosynthesis the mature TGF-xcex2 dimer is cleaved from the propeptide dimer. TGF-xcex2 latency results in part from the non-covalent association of propeptide and mature TGF-xcex2 dimers (Pircher et al., 1984, 1986; Wakefield et al., 1987; Millan et al., 1992; Miyazono and Heldin, 1989). Consequently, the propeptide dimer is often referred to as the latency associated protein (LAP), and LAP plus the disulfide-bonded TGF-xcex2 dimer are also known as the small latent complex. In the extracellular space small latent complexes must be dissociated to activate mature TGF-xcex2. The mechanism of activation of the latent complex is thought to be one of the most important steps governing TGF-xcex2 effects (Lyons et al., 1988; Antonelli-Orlidge et al., 1989; Twardzik et al., 1990; Sato et al., 1993).
In certain lines of cultured cells small latent growth factor complexes may contain additional high molecular weight proteins. The best characterized of these high molecular weight proteins is the latent TGF-xcex2 binding protein, or LTBP (Miyazono et al., 1988; Kanzaki et al., 1990; Tsuji et al., 1990; Olofsson et al., 1992; Taketazu et al., 1994). LTBP produced by different cell types is heterogeneous in size, perhaps because of alternative splicing or because of tissue-specific proteolytic processing (Miyazono et al., 1988; Wakefield et al., 1988; Kanzaki et al., 1990; Tsuji et al., 1990). Latent TGF-xcex2 complexes that contain LTBP are known as large latent complexes. LTBP has no known covalent linkage to mature TGF-xcex2, but rather it is linked by a disulfide bond to LAP.
Regarding the novel protein LTBP-2 or LTBP-3, the present invention concerns DNA segments, that can be isolated from virtually any mammalian source, that are free from total genomic DNA and that encode proteins having LrBP-2-like or LTBP-3-like activity. DNA segments encoding LTBP-2-like or LTBP-3-like species may prove to encode proteins, polypeptides, subunits, functional domains, and the like.
As used herein, the term xe2x80x9cDNA segmentxe2x80x9d refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding LTBP-2 or LTBP-3 refers to a DNA segment that contains LTBP-2 or LTBP-3 coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term xe2x80x9cDNA segmentxe2x80x9d, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified LTBP-2 or LTBP-3 gene refers to a DNA segment including LTBP-2 or LTBP-3 coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term xe2x80x9cgenexe2x80x9d is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides.
xe2x80x9cIsolated substantially away from other coding sequencesxe2x80x9d means that the gene of interest, in this case, a gene encoding LTBP-2 or LTBP-3, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode an LTBP-2 or LTBP-3 species that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:2 or SEQ ID NO:4. In other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that include within their sequence a nucleotide sequence essentially as set forth in SEQ ID NO:1 or SEQ ID NO:3.
The term xe2x80x9ca sequence essentially as set forth in SEQ ID NO:2 or SEQ ID NO:4xe2x80x9d means that the sequence substantially corresponds to a portion of SEQ ID NO:2 or SEQ ID NO:4 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2 or SEQ ID NO:4. The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein (for example, see section 7, preferred embodiments). Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO:2 or SEQ ID NO:4 will be sequences that are xe2x80x9cessentially as set forth in SEQ ID NO:2 or SEQ ID NO:4xe2x80x9d.
In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:1 or SEQ ID NO:3. The term xe2x80x9cessentially as set forth in SEQ ID NO:1 or SEQ ID NO:3xe2x80x9d is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:1 or SEQ ID NO:3 and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO:1 or SEQ ID NO:3. Again, DNA segments that encode proteins exhibiting LTBP-2-like or LTBP-3-like activity will be most preferred.
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5xe2x80x2 or 3xe2x80x2 sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5xe2x80x2 or 3xe2x80x2 portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3. Nucleic acid sequences that are xe2x80x9ccomplementaryxe2x80x9d are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term xe2x80x9ccomplementary sequencesxe2x80x9d means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:1 or SEQ ID NO:3, under relatively stringent conditions such as those described herein.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to SEQ ID NO:1 or SEQ ID NO:3, such as about 14 nucleotides, and that are up to about 10,000 or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases. DNA segments with total lengths of about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful.
It will be readily understood that xe2x80x9cintermediate lengthsxe2x80x9d, in these contexts, means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001, 13,002 and the like.
It will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NO:1 or SEQ ID NO:3; and SEQ ID NO:2 or SEQ ID NO:4, respectfully. Recombinant vectors and isolated DNA segments may therefore variously include the LTBP-2 or LTBP-3 coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include LTBP-2 or LTBP-3-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
The DNA segments of the present invention encompass biologically functional equivalent LTBP-2 or LTBP-3 proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.
If desired, one may also prepare fusion proteins and peptides, e.g., where the LTBP-2 or LTBP-3 coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a LTBP-2 or LTBP-3 gene, as may be obtained by isolating the 5xe2x80x2 non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR(trademark) technology, in connection with the compositions disclosed herein.
In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with an LTBP-2 or LTBP-3 gene in its natural environment. Such promoters may include LTBP-2 or LTBP-3 promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).
In connection with expression embodiments to prepare recombinant LTBP-2 or LTBP-3 proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire LTBP-2 or LTBP-3 protein or functional domains, subunits, etc. being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of LTBP-2 or LTBP-3 peptides or epitopic core regions, such as may be used to generate anti-LTBP-2 or LTBP-3 antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 15 to about 50 amino acids in length, or more preferably, from about 15 to about 30 amino acids in length are contemplated to be particularly useful.
The LTBP-2 or LTBP-3 gene and DNA segments may also be used in connection with somatic expression in an animal or in the creation of a transgenic animal. Again, in such embodiments, the use of a recombinant vector that directs the expression of the full length or active LTBP-2 or LTBP-3 protein is particularly contemplated.
In addition to their use in directing the expression of the LTBP-2 or LTBP-3 protein, the nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the some sequence as, or is complementary to, a 14 nucleotide long contiguous sequence of SEQ ID NO:1 or SEQ ID NO:3 will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to LTBP-2 or LTBP-3-encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so, identical or complementary to SEQ ID NO:1 or SEQ ID NO:3, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow LTBP-2 or LTBP-3 structural or regulatory genes to be analyzed, both in diverse cell types and also in various mammalian cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 10-14 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
The use of a hybridization probe of about 10-14 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 10 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 contiguous nucleotides, or even longer where desired.
Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 and to select any continuous portion of the sequence, from about 10-14 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors, such as, by way of example only, one may wish to employ primers from towards the termini of the total sequence.
The process of selecting and preparing a nucleic acid segment that includes a contiguous sequence from within SEQ ID NO:1 or SEQ ID NO:3 may alternatively be described as preparing a nucleic acid fragment. Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR(trademark) technology of U.S. Pat. No. 4,603,102 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of LTBP-2 or LTBP-3 gene or cDNA fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to forms the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of 50xc2x0 C. to 70xc2x0 C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating LTBP-2 or LTBP-3 genes.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate LTBP-2 or LTBP-2 or LTBP-3-encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from 20xc2x0 C. to 55xc2x0 C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.